Recently, S1P and S1P receptors have been specifically targeted as treatments for immune-mediated diseases, particularly the immunosuppressant FTY720 (Fingolimod Gilenya), an S1P analogue that has recently become the first oral therapy for relapsing Multiple Sclerosis. FTY720 effectively decreases relapse rates better than interferon ? therapy; however, a subset of patients treated with FTY720 develop very severe relapses and even tumorfactive lesions. This finding suggests that S1P1-mediated signaling may be involved in some unknown immune regulation, in addition to its role in T and B cell trafficking. Although it remains controversial whether FTY720, after being phosphorylated to FTY720-P in vivo, acts as an agonist or a functional antagonist, understanding how FTY720 affects the function of T regulatory cells (Tregs) is crucial for limiting the adverse effects of lng term therapy with this drug. In order to address the specific role of S1P on Tregs, we have generated mice that lack S1P1 only in Tregs. We have found 10 times more Tregs in the thymus of S1P1 Treg-deficient mice compared to WT mice In the periphery however, we have found that S1P1 is not required for the egress of Tregs from lymph nodes or their tissue trafficking. Tregs that lack S1P1 could still suppress in vitro and expressed normal levels of IL-10, TGF beta, CD25 and CTL4; however, these Tregs lost their ability to control immune responses in vivo. Indeed, S1P1 Treg-deficient mice developed rapid and severe systemic autoimmunity, revealing a critical role of S1P for the function of Treg. Thus, from our preliminary data we hypothesize that S1P1-mediated signaling is: 1) crucial for the egress of Tregs from the thymus, 2) crucial for the fine positioning of Tregs within lymphoid organs and 3) crucial for the suppression function of Tregs in tissues such as the CNS. Whereas S1P1-mediated signaling is required for T cell egress and trafficking, S1P1 may control the functions of Tregs differently. To address these hypotheses, we propose to carry out the following specific aims: Aim 1. Role of S1P1 in the generation of Tregs in the Thymus Aim 2. Investigate whether compromised S1P1 expression affects Treg positioning in secondary lymphoid tissues Aim 3. Determine the requirement of selective S1P1 for the migration of Treg cells in the CNS and the development of EAE. This novel mouse strain and new findings will help us understand how S1P and Fingolimod affect the function of Tregs during the course of Multiple Sclerosis.